title: Fluorescence enhancement upon G-quadruplex folding: synthesis, structure, and biophysical characterization of a dansyl/cyclodextrin-tagged thrombin binding aptamer.
abstract: A novel fluorescent thrombin binding aptamer (TBA), conjugated with the environmentally sensitive dansyl probe at the 3'-end and a β-cyclodextrin residue at the 5'-end, has been efficiently synthesized exploiting Cu(I)-catalyzed azide-alkyne cycloaddition procedures. Its conformation and stability in solution have been studied by an integrated approach, combining in-depth NMR, CD, fluorescence, and DSC studies. ITC measurements have allowed us to analyze in detail its interaction with human thrombin. All the collected data show that this bis-conjugated aptamer fully retains its G-quadruplex formation ability and thrombin recognition properties, with the terminal appendages only marginally interfering with the conformational behavior of TBA. Folding of this modified aptamer into the chairlike, antiparallel G-quadruplex structure, promoted by K(+) and/or thrombin binding, typical of TBA, is associated with a net fluorescence enhancement, due to encapsulation of dansyl, attached at the 3'-end, into the apolar cavity of the β-cyclodextrin at the 5'-end. Overall, the structural characterization of this novel, bis-conjugated TBA fully demonstrates its potential as a diagnostic tool for thrombin recognition, also providing a useful basis for the design of suitable aptamer-based devices for theranostic applications, allowing simultaneously both detection and inhibition or modulation of the thrombin activity.
authors.affiliation: Department of Pharmacy, University of Naples Federico II , via D. Montesano 49, I-80131 Naples, Italy.
authors.forename: Stefano
authors.initials: S
authors.lastname: De Tito
authors.forename: François
authors.initials: F
authors.lastname: Morvan
authors.forename: Albert
authors.initials: A
authors.lastname: Meyer
authors.forename: Jean-Jacques
authors.initials: JJ
authors.lastname: Vasseur
authors.forename: Annunziata
authors.initials: A
authors.lastname: Cummaro
authors.forename: Luigi
authors.initials: L
authors.lastname: Petraccone
authors.forename: Bruno
authors.initials: B
authors.lastname: Pagano
authors.forename: Ettore
authors.initials: E
authors.lastname: Novellino
authors.forename: Antonio
authors.initials: A
authors.lastname: Randazzo
authors.forename: Concetta
authors.initials: C
authors.lastname: Giancola
authors.forename: Daniela
authors.initials: D
authors.lastname: Montesarchio
mesh_terms: D052157:Aptamers, Nucleotide / Q000737:chemistry*; D004247:DNA / Q000737:chemistry*; D003619:Dansyl Compounds / Q000737:chemistry*; D005453:Fluorescence; D054856:G-Quadruplexes; D006801:Humans; D008958:Models, Molecular; D015394:Molecular Structure; D009690:Nucleic Acid Conformation; D047392:beta-Cyclodextrins / Q000737:chemistry*
chemical_list: D052157:Aptamers, Nucleotide; D003619:Dansyl Compounds; D047392:beta-Cyclodextrins; C081676:thrombin aptamer; D004247:DNA; C031215:betadex